Vitronectin Destroyed Intestinal Epithelial Cell Differentiation through Activation of PDE4-Mediated Ferroptosis in Inflammatory Bowel Disease

Figure 4

VTN modulated PDE4/PKA/CREB signaling. (a) Immunofluorescence was employed to display the CREB nuclear localization in CaCO₂ cells treated with VTN or VTN combined with roflumilast (Rofl) for 1 hr. (b) CaCO₂ cells were serum starved for 24 hr and then stimulated as indicated for an additional 1 hr. Nuclear and cytosolic CREB levels were determined by western blotting. GAPDH and lamin A/B were served as internal controls for the cytosolic and nuclear fractions, respectively. (c) After serum starved for 24 hr, CaCO₂ and HT-29 cells were treated with or without VTN for 1 hr, the total proteins were collected and separated to detect indicated protein by western blotting. α-Tubulin was taken as internal control. (d) HT-29 cells were serum starved for 24 hr after 80% confluence, then stimulated as indicated for 1 hr. Immunoprecipitated (IP) was employed to analyze the interaction between PKA and CREB in response to VTN and VTN combined with Rofl. (e) After serum starved for 24 hr, the total cell was pretreated with Rofl for 1 hr; subsequently, followed by stimulation with or without VTN for further 48 hr, the total protein was collected to detect indicated protein. α-tubulin was served as internal control. (f) CaCO₂ cells were treated with basic medium for 24 hr, followed by stimulation with VTN and VTN combined with Rofl for further 48 hr, the level of ROS was detected. (g) Intestinal organoids assay was performed to analyze the effect of VTN-Rofl on cell differentiation. (h) After transfection with indicated siRNA for 24 hr, HT-29 cells were starved for 12 hr and treated with or without VTN for another 24 hr; the total protein was collected to detect indicated proteins.

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